The present invention is generally in the field of biological formulations, in particular, methods for making stable and useful fungal conidial formulations.
A number of investigators have explored the possibility of using entomopathogenic fungi for insect biocontrol. The potential for successful application of entomopathogenic fungi as biological control agents has only recently begun to be developed. The successful commercial use of the biocontrol agents has required significant advances in a number of fundamental research areas including strain selection, bioassay procedures, process development, and in production related areas such as manufacturing, packaging and distribution.
The entomopathogenic fungi classified as Hyphomycetes are natural insect pathogens which have a biphasic biological cycle consisting of a mycelial vegetative phase and an asexual conidiospore reproductive phase. Since only the reproductive propagules, the conidia, are involved in the infective process, the conidia are the preferred material for use in biocontrol. The conidia infect a susceptible insect host through the integument. Three steps have been recognized in the development of the infection:
a) adhesion of the spore to the insect cuticle; PA1 b) formation and penetration of the insect cuticle by the germ tube; and PA1 c) growth of the fungus within the insect body.
In one preferred method of insect control with entomopathogenic fungi, the fungus is applied in the form of a conidial dispersion or suspension. In another, the treatment involves storage and distribution of conidia in a chamber which is attractive to the insects. Problems are inherent in all methods of conidial application and the lack of suitable formulations and satisfactory delivery systems have not yet been fully overcome, limiting the large scale development of fungi as biological control agents.
In nature, the initial fungus-host interaction is by chance. The conidiospores of fungi such as Metarhizium anisopliae and Beauveria bassiana, among others, have evolved spore surface (or coat) physicochemical attributes which enhance and probably mediate the attachment of the spore to the insect cuticle. No pesticidal activity is possible in the absence of attachment and germination. Therefore, the role of process components which might affect attachment and germination by the conidia must be considered during the selection of the methodologies that might be used in any commercial development of fungal biocontrols. Any process or agent which adversely interferes with either the attachment or germination steps could render the conidia noninfective and therefore unacceptable for use in biocontrol applications.
In addition to good insecticidal activity, essential features of an acceptable fungal biological control product include extended shelf life, under a variety of temperature conditions, and adaptability to a variety of targeted applications. The instability of the fungal conidia in suspension can result in a loss of viability, or, if the conidia germinate, difficulty in spraying the conidia, as well as a loss of insecticidal activity.
The inability heretofore to successfully manipulate and control the microenvironment of the spore has severely limited the applicability of such conidia for commercial use. The lack of acceptable materials that do not adversely interfere with or suppress viability, attachment, or germination has been a major obstacle to the development of fungal-based biological control systems utilizing entomopathogenic fungi.
The conidia of many entomopathogenic fungi by virtue of their hydrophobicity are refractory to mixing and dispersion in aqueous diluents. Clearly, the development of stable, efficacious, water-based fungal biocontrol products with broad spectrum activity and extended shelf-life has been almost nonexistent. Many of these problems are a result of inadequacies of the current formulation technology with regard to any biocontrol agents, particularly fungal agents.
Surfactants, wetting agents and related compounds are known to facilitate the production of monodisperse suspensions of inert particles. In most nonbiological systems these additives are routinely employed to facilitate dispersion. In nonbiological systems, attributes such as performance, cost and application dictate selection. Moreover, even with biologics such as proteins, carbohydrates, lipids and mixtures where viability is not an issue, many different classes and concentrations of the agents can yield usable, functional commercial products. In contrast, living microorganisms require a very stringent and precise set of selection conditions where maintenance of viability, a preferred physiological state and functional biological activity are key requirements to be met. As a result of the unique needs inherent in living biological systems, few surfactants, wetting agents and related materials are known which perform satisfactorily, especially for commercial applications. Long term storage, maintenance of viability and high virulence are but a few of the required attributes for a commercial biological control product which have not been attainable heretofore with the known surfactants. Specifically, surfactants in general interfere with attachment and germination of conidia, and no stable aqueous formulations of conidia and surfactant are known since many fungi such as M. anisopliae and B. bassiana metabolize surfactants, detergents, wetting agents and similar materials. Within as brief a period as 72 hours, an aqueous suspension of conidia in the presence of known surfactants will germinate and grow to form a mycelial mass which is useless for commercial insect control.
In toto, the lack of usable surfactants, wetting agents and similar functional materials compatible with viable microorganisms such as entomopathogenic fungi has negatively impacted areas as diverse as laboratory research (assay development), manufacturing (processing aids), formulations and commercial applications. The concentrations of surface active agents which have been described in the literature as components of conidial suspensions has not exceeded 0.5 percent (volume to volume for liquids; weight to volume for solids), and the reported duration of exposure has been limited to just a few hours. Longer exposure results in either loss of viability or germination and growth of the conidia to form the vegetative mycelial morphology.
It is therefore an object of the present invention to provide materials useful in producing aqueous suspensions of viable, monodispersed conidia with pesticidal activity, stable to both long and short term storage.
It is a still further object of this invention to provide a conidia-compatible wetting agent for use in dry conidial formulations such as wettable (water-dispersible) granular powders and other formulations where a substantially water-based conidial suspension is required.
It is another object of the present invention to provide materials that exhibit antibacterial, but not antifungal activity, which do not inhibit germination after application or otherwise alter conidial viability, that are useful in preserving condia.
It is still another object of the present invention to provide materials that are useful in manipulating and controlling conidial viability and propagation.